The human immunodeficiency virus (HIV) is recognized to be the agent resposible for acquired immunodeficiency disease syndrome (AIDS). HIV is a lentivirus. The processing of HIV-fusion polypeptides, the products of the gag and pol genes of HIV, is carried out by a virally-encoded protease. This processing has been demonstrated to be crucial to the replication of both HIV type-1 (HIV-1) and HIV type-2 (HIV-2), collectively referred to herein as HIV.
Inhibition of the viral protease and search for its inhibitors have been the subjects of immense interest for the last few years. Kinetic assays for HIV protease include HPLC assays, cleavage of a radiolabeled decapeptide, and spectrophotometric assays utilizing chromogenic and fluorogeneic substrates. HPLC assays, compared with spectrophotometric assays, are time-consuming and discontinuous. Other assay techniques are cumbersome and require synthesis of appropriate substrates. Conventional wisdom dictates that none of these assays would allow high-volume evaluation of protease inhibitors in animal models.
There is a need for a rapid, sensitive, generic, and high volume assay for the HIV protease for in vitro screening as well as for tracking its inhibitors in the blood in common laboratory animals. Therefore, it is conceived that if, in fact, one could find a substrate for the HIV protease that can be cleaved by the protease to produce a target for specific antibodies that will not cross react with the uncleaved substrate, such a system would be valuable for testing HIV protease inhibitors in animal models and in humans.
In addition, there is a need for sensitive, i.e, accurate and precise, diagnostic kits to determine whether or not a patient has been exposed to HIV. The most common method currently being exploited is the use of anti-HIV antibody immunoassay kits. While these kits are easy to use and relatively inexpensive, there is a great concern about the high number of false positives inherent in such methodology. Alternatively, a sample taken from a patient is prepared and separated on a Western blot to visually identify the presence of HIV protease using anti-HIV antibodies. This methodology, although very accurate, is time consuming and expensive. There is a need for a simple, inexpensive and highly sensitive method to detect the presence of antibodies to HIV proteins.
The present invention relates to a peptide substrate that contains an amino acid sequence capable of being cleaved by HIV protease and that can be distinguished from the fragments generated by protease cleavage of the substrate. The substrates of the present invention are useful in the screening and study of compounds which inhibit HIV protease and in diagnostic kits to detect the presence of antibodies against HIV protease.
The present invention provides an assay of identifying compounds useful as HIV protease inhibitors which can be used to investigate compounds in vitro or from fluids taken from patients. By providing a substrate comprising an HIV protease cleavage site and by having the ability to distinguish cleaved product from uncleaved substrate, the present invention provides a method of testing the inhibitory activity of compounds. The present invention provides a system for high volume screening of compounds which is used to identify those compounds useful as HIV protease inhibitors. The same system is particularly useful in bioassays to track activity of inhibitor compounds in vivo. There is a need for an easy and inexpensive diagnostic test system which can detect the presence of antibodies against HIV proteins accurately. Therefore, it is conceived that if one could find a substrate for the HIV protease that can be cleaved by the protease to produce a target for specific antibodies that will not cross react with the uncleaved substrate, such a system would be valuable for detecting the presence of antibodies against HIV protease in a biofluid sample.
The present invention provides an assay for identifying the presence of antibodies to HIV protease in fluids taken from patients; such an assay being used to diagnose HIV infection. By combining an HIV protease cleavage site on a substrate with the ability to distinguish cleaved product from uncleaved substrate, the present invention provides the ability to detect the presence of antibodies against HIV protease. The present invention provides a system for accurate detection of antibodies against HIV protease simply and inexpensively.